Previously, we demonstrated a novel molecular mechanism for Akt activation where Akt interaction with PS-binding residues, particularly in the regulatory domain, is critical for Akt phosphorylation at S473 by mTORC2. Phosphorylation at S473 is an important modulator of Akt activation, which can serve as a new target different from well-recognized PIP3- or ATP-binding for drug development. As an effort to identify Akt inhibitors based on this new target, we developed a cell-based high throughput screening (HTS) assay using homogeneous time-resolved fluorescence (HTRF) detection. We subsequently tested approximately 400,000 MLSCN (Molecular Libraries Screening Center Network) compound collections in collaboration with NCGC. Among approximately 1800 compounds screened as positive hits, about 100 compounds have been selected for tertiary assays during this period. The small molecules that inhibit specifically Akt S473 phosphorylation/mTORC2 activity thus identified will not only serve as valuable research tools but also may have significant therapeutic potential with fewer side effects, especially for conditions involving hyperactive Akt signaling such as cancer and Alzheimers disease. As PS accumulation is an important aspect of DHA- or ethanol-mediated effects on Akt signaling, we continued to characterize the neuronal PS synthesis. While it is postulated that the conformational status of phosphatidylserine synthases (PSS) is intimately coupled to its activity, the crystal structure of this integral membrane protein is not available at present. To provide molecular insight for the observed functional selectivity of PSS towards DHA-containing phospholipids, we probed the conformation of PSS2 using chemical crosslinking and mass spectrometry. Immunopurified PSS2 was incubated with disuccinimidyl suberate (DSS), a lysine specific crosslinker. Monomeric or dimeric protein bands were separated by SDS-PAGE and subjected to tryptic digestion. Crosslinked peptides were analyzed by nanoLC-ESI-MS/MS. Several crosslinked lysine pairs, including K267-K252, K265-K284, K265-249, and K265-351, K249-K267 and K419-K425, were identified within PSS2 molecules. As the crosslinked lysine pairs are located within 24 defined by DSS, the data provided for the first time the spatial distance information in the PSS2 structure. Conformational changes of PSS2 monitored by the crosslinking profile may provide insight on specificity of this enzyme towards DHA-containing species as well as ethanol-induced modulation of PSS2 activity. We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant mechanism for hippocampal neuritogenesis, synaptogensis and glutamatergic synaptic activity. During this period, we investigated signaling mechanisms for synaptamide-induced neuritogenesis using cortical neurons. We found that 10-100nM synaptamide significantly promote dendrite and axon growth in cortical cultures. In addition, synaptamide reduced the size of axonal varicosities, structures involved in axon morphogenesis and injury, which may represent facilitated transport of axonal components for axonal growth. Kinome search for the phosphorylation events, cell-based dual luciferase CRE reporter assay as well as western blot analysis consistently indicated that the cAMP/PKA/CREB pathway is involved in synaptamide-induced neuritogenesis. We continued our investigation on the differentiation of neural stem cells (NSCs) obtained from E14.5 rat embryos and found that synaptamide, which is produced from DHA by NSCs, induces neuronal differentiation of NSCs at low nM concentrations. This neurogenic property of synaptamide was abolished or significantly diminished by PKA inhibitors or PKA knock down, while suppression of fatty acid amide hydrolase increased the neurogenic capacity of DHA or synaptamide. From these results we conclude that synaptamide is a potent DHA-derived mediator for neurogenic differentiation of NSCs acting through PKA/CREB activation. To understand the role of DHA in aging at the molecular level, we investigated during this period the role of DHA in age-related regulation of synaptic proteome using a strategy involving synaptic plasma membrane (SPM) preparation, SDS PAGE, in-gel tryptic digestion and nanoLC-ESI-MS/MS analysis. Relative quantification was performed by either a label-free approach or differential labeling with 16O/ 18O water. The synaptic proteins differentially expressed in young (4 month old) and aged (15 month old) mouse brains was identified by mass spectrometry and validated by western blot or mRNA analysis. We found significant reduction of 17 proteins in aging brains including glutamate receptors (NMDA subunit 2 and AMPA2), munc18-1, PSD-95, sv2b, dynamin-1, VAMP2 and SNAP25, septin-3, neuroplastin, glutamate transporters (excitatory amino acid transporter1 and vesicular glutamate transporter 1), fodrin-alpha, rab3A, neuroplastin, SNAP-alpha and synaptopodin. Among these, PSD-95, synaptopodin, SV2b, SNAP25, SNAP-alpha, NR2b, AMPA2 receptor and fodrin-alpha were found to be further down-regulated with aging under DHA-deprived conditions. These data indicate that DHA plays an important role in aging by preserving the status of some of the neurotransmission-regulating synaptic proteins. We continued to develop an approach for identification of novel metabolites derived from uniformly 13C-labeled substrates using HPLC/ESI-MS/MS and software-assisted peak finding routine. Using platelet and leukocyte metabolites formed from 13C-arachidonic acid (AA) and DHA as model systems. Based on the unique isotope profile of 13C-AA- or 13C DHA-derived metabolites together with the predicted mass differences from the corresponding 12C-AA- or DHA-counterparts, an automatic peak finding algorithm has been developed and its applicability is being tested. Once confirmed, this metabolite search strategy will be extended to other classes of bioactive compounds for which uniformly labeled 13C-isotopes are available. During this period, we have also initiated research to probe conformation of mu-opioid receptor (MOR) which is known to exert biological effects such as analgesia and physical dependence. Despite recent breakthrough in X-ray crystallographic studies of ligand-bound opioid receptors, detailed structural changes and molecular mechanisms responsible for agonist-selective receptor desensitization and internalization are not fully understood. We established a new strategy to probe the receptor activity in relation to conformation of MOR in a physiologically relevant setting. Flag-myc-MOR was expressed in Neuro 2A cells, chemically crosslinked in intact cells with lysine-specific crosslinkers with different spacer arm length, and DAMGO-induced MOR phosphorylation was monitored. We found that MOR phosphorylation was inhibited when MOR was modified with longer crosslinkers (24 ) but not with a shorter crosslinker (19 ) prior to DAMGO stimulation, indicating that a subtle restriction of the MOR structural profile prevents activation of this receptor. To probe the conformational status of MOR, the Flag-myc-MOR expressed in Neuro 2A cells was crosslinked in intact cells, immunopurified and subjected to SDS-PAGE, chymotryptic digestion and nanoLC-ESI-MS/MS. Our preliminary data revealed a cross-linked lysine pair (K211-K235) within the extracellular loop 2 (EL2), in addition to an inter-EL linking between EL2 and EL3 (K235-K305) in the basal state. We will subsequently investigate the agonist specific alteration of extracellular loop arrangements. Once the structural changes are probed, we will investigate the influence of ethanol and membrane lipid matrix in relation to desensitization properties.